Quantitative evaluation of anti-FOXE-3 Ab identifies a subset of anti-CENP positive systemic sclerosis patients who are less prone to develop active disease

Background and objective: In a subset of systemic sclerosis (SSc) patients (pts), anti-CENP-A Ab cross reacts with a FOXE-3-derived peptide (FOXE3p), which presents a striking homology with one (Ap17-30) of the 2 immunodominant epitopes of CENP-A. Preliminary analysis, obtained by absorbance binding, showed that active disease was less likely to occur in anti- FOXE-3+ patients as compared to anti- FOXE-3- patients. Here, we have set up a titration assay using appropriate standards to quantify IgG anti-FOXE-3 and to define whether any clinical correlate exist with the presence of anti FOXE3. As control, clinical correlates with the level of antibodies to Ap17-30 and to the 2nd immunodominant epitope of CENP-A (spanning amino acids 1-to 17, Ap1-17) were also evaluated. Methods: Ap17-30, FOXE3p and Ap1-17 were synthesized at Primm peptide synthesis service (Naples, Italy). The reactivity of serum IgG to specific peptides was measured by indirect ELISAs, using BSA-conjugated peptide as coating reagents and HRP-conjugated xeno-Ab to the Fc portion of human IgG as probe. Sera from 117 SSc pts were tested for anti-centromere and anti-topoisomerase Ab (Diamedix). Calibration curves to measure the levels of Ab were settled, using IgG from sera purified by affinity column coupled to Ap17-30 and Ap1-17 peptides. Clinical records were reviewed to achieve demographic data and information about organ involvement and disease activity, previously evaluated according to a core set of 10 variables. Control sera were obtained from SLE (#47 pts) and from healthy blood donor (HBD) (#25 pts). Results: Purify IgG anti-Ap1-17 and anti-Ap17-30 were used to set-up a standard calibration curve to measure serum levels of anti-Ap1-17 (detection limit range: 2.68-6037ug/ml) and Ap17-30 (0.36-32.85 ug/ml) respectively. As anti-Ap17-30 cross-react with FOXE-3, calibration curve to measure serum levels of Abs to this peptide was achieved using purified anti-Ap17-30 IgG and FOXE-3p as coating reagents (detection limit range: 9.25-880 ug/ml). Of 117 SSc sera, 68 (58.1%) were anti-CENP positive (CENP+) and 94% (#65) of them reacted with Ap17-30 and/or Ap1-17, while no reactivity was observed on the replacement of CENP+ with CENP-, SLE, and HBD sera. The best cut-off concentration of Ab levels able to discriminate CENP+ from CENP- patients (obtained with ROC analysis) were >10.4 ug/ml (sensitivity: 86.8, specificity 98%), >1.2ug/ml (sens. 83%, spec. 96%) and > 74.5 ug/ml (sens.>52.6%, spec. 90%) for anti-Ap1-17, anti-Ap17-30 and anti-FOXE3p respectively. Following dichotomization of CENP+ patients according to their disease activity index score in group 1 (61 patients with score <3), and group 2 (7 patients with score> 3; active disease), a Fisher exact test showed a statistical association of FOXE3p+ sera with group 1 patients (p=0.003, OR: 39.5). Conclusion: Our data confirm the identification of a subset of patients (FOXE3+) that are less likely to have active disease.